Schizosaccharomyces pombe as a predictor toxicity tool

The fission yeast Schizosaccharomyces pombe is frequently used as a genetically manipulable model system, offering valuable understandings into cellular mechanisms. In the present study, a comprehensive step-by-step methodology for the research of the action mechanisms and detoxification by efflux pumps is showed. The protocol involves the thawing and culture of yeast cells in liquid medium under controlled conditions to ensure exponential growth. After that, a dose-response assessment is carried out by culturing wild-type cells in liquid medium, followed by exposure to increasing concentrations of the toxic substances. Optical density measurements are taken spectrophotometrically after exposure, and the process is repeated at least three times for quantitative analysis. Subsequently, defective mutants are selected to explore specific mechanisms of action or detoxification by efflux pumps, with cultures prepared and treated similarly to the wild type. Optical density measurements are again taken after exposure for quantitative analysis. This methodology ensures robust and reproducible results for the research toxic substances effects on S. pombe.- Schizosaccharomyces pombe is an adequate tool to evaluate contaminants toxicity.- Dose-responses curves are obtained on wild type to evaluate toxicity mechanisms.- This methodology ensures robust and reproducible results for the research toxic substances effects on S. pombe.


Background
The fission yeast S. pombe is a eukaryotic ideal model organism for discovering chemical inhibitors and analyzing drug mechanisms of action by studying its defective strains [ 8 ].For all of that, it has been decided to search for a method that provide us relevant information in a few hours about cytotoxicity, action mechanisms and pumps detoxication generated by several daily drugs.Here, it has been proposed a procedure based on two differentiated steps.In the first one, a dose-response assessment is completed in the wild type strain and in the second one, the investigation of the main mechanisms of action or detoxification by efflux pumps is carried out in the defective strains ( Fig. 3 ).

Key resources TABLE
The S. pombe strains were provided from the Centro Andaluz de Biología del Desarrollo (CABD), from R.R. Daga´s and S. Moreno´s labs ( Tables 1 -3 ).Extremely marked specificity for oxidative stress and very high sensitivity to osmotic stress.Similar sensitivity as sty1 Δ, but with some components due to pmk1 deletion [ 5 ] rad3 Δ Extremely marked specificity for DNA damage.Very high sensitivity for oxidative stress [ 7 ] Step-by-step method details A. Preparing the experimental model 1. S. pombe yeast cells are thawed on agar plates and incubated in a stove for 48 h at 25 °C to initiate their growth.2. A pre-inoculum is prepared by taking a small amount of biomass from the plate and putting it into 5 mL of YES medium in a sterile flask with a capacity of 25 mL.1 3. The flask with the yeast pre-inoculum is cultured for at least two generation times (2-3 h) with continuous shaking at 180 rpm in an incubator shaker at 25 °C.4. The optical density is measured at 595 nm (or the cells counted) by adding 100 μL in a well of a 96-well microplate to assure the correct growth (about 0.08-0.1).The culture is diluted in YES about 1:4 if the time to growth is 8 h and about 1:10 if the time is 16 h.5.This process must be repeated at least two times before starting the assay. 2.Dose-response assessment 6.The optical density is measured, and the culture is diluted to 0.004 OD with a final volume of 55 mL in a 250 mL flask.7. The flask is shaken in the incubator shaker at 180 rpm and 25 °C for 15 min.8.The content of the flask is divided into 10 flasks, each containing 5 mL of YES. 9. To the first flask, drug is not added as it will serve as a negative untreated control group.10.Increasing concentrations of the toxic substance under study are added to flasks 2 through 10, maintaining the same dilution /solvent concentration. 3".11.The flasks are shaken in the incubator shaker at 180 rpm and 25 °C for 15 min.12.The content of the flasks is transferred to a 96-well plate.Each well B-F is filled with 200 μL of the corresponding exposure solutions (6 wells per concentration).Thus, column 1 is filled with YES medium and it serves as the blank control, column 2 is filled with the content of flask 1, column 3 with flask 2, and so on until column 10.The remaining wells along the edges should be filled with YES or distilled water to maintain humidity.13.Place the 96-well plate under constant agitation at 1200 rpm on a plate shaker for 16 h at 25 °C in a humidity chamber.14.After the period of exposure, the density of the cell populations is measured spectrophotometrically using a microplate reader Varioskan Flash4 ( Fig. 1 ).15.Each experiment is repeated at least three times.16.Quantitative analysis of the values is performed, considering that OD values are correlated to the number of S. pombe cells.
Experimental groups are statistically compared to the untreated cells.17.A dose-response regression is represented with Excel or other software [ 1 , 2 ].

C. Investigation of the main mechanisms of action and detoxication by efflux pumps
18.After the dose response analysis, two concentrations that showed from 20 to 50 % growth inhibition in the wild-type strain are selected (EC80, EC50) to research the main mechanisms of action of the chemical or the detoxication by efflux pumps.
Depending on the type of study, specific defective strains should be select ( Tables 4 and 5 ).19.Once the defective mutants are selected, yeast are cultured at least two times to exponential growth after the pre-inoculum.
The process is also carried out with the wild type.

Table 5
Strains used for the study of detoxication by efflux pumps.20.Subsequently, the OD is determined, and the cultures are diluted to 0.004 OD, reaching a final volume of 18 mL in a 100 mL capacity flask for each strain.21.The flasks are agitated in the incubator shaker at 180 rpm and 25 °C for 15 min.22.The contents of the flasks are distributed into three separate flasks for each strain, containing each one 5 mL.23.In the first flask, no drug is added, as it is the negative untreated control.In the second, it was added concentration 1 (C1) and in the third, concentration 2 (C2) of the selected toxic.This process is repeated with the wild type strain and all the selected mutants.24.All the flasks are shaked 15 min in the incubator shaker at 180 rpm at 25 °C.25.The 96-well plate is filled with the contents of each flask, adding 200 μL/well starting from the wild type (WT) in the column 2, followed by WT + C1 in the column 3, WT + C2 in the column 4, and continuing with the mutants in the same order, respectively.Column 1 is filled with YES, functioning as the blank/solvent control.The remaining wells along the edges can be filled with YES or distilled water to avoid interferences.The experiments are conducted in triplicate.26.The 96-well plate is placed on a plate shaker and agitated constantly at 1200 rpm for 16 h at 25 °C in a humidity chamber.27.After 16 h exposure period, the density of the cell population is measured spectrophotometrically5 ( Fig. 2 ).28.The analysis of the results is conducted, considering that OD values correlate with the number of S. pombe cells.Experimental groups are statistically compared to the untreated cells of each tested concentration.29.A dose-response regression is represented with Excel or other software [ 1 , 2 ].

Method validation
For the dose-response assessment, the kind of graphic representation obtained follows the subsequent pattern in which the percentage of proliferation is represented on the x axis and concentration on the y axis in logarithmic scale, previous published in Álvarez-Herrera et al. [ 1 ].
In the case of the investigation of the main mechanism of action, triclosan has been selected as an example due to its high reproducibility.In the x axis shows the percentage of proliferation and the y axis, the concentration of triclosan selected, also previous published in Álvarez-Herrera et al. [ 2 ].
The graphic representation in the investigation of detoxication by efflux pumps is similar to Fig. 4 and the only difference is the defective mutants used.

Limitations
Two important aspects should to be taking into account to obtain reproducible data: -It must be very precise in the exposure and measurement times, due to after the proposed time (16 h) the yeasts enter in the stationary phase.-It is very important that the agitation on the plate-shaker was constantly at 1200 rpm.

Fig. 1 .
Fig. 1.Dose response assessment.Schematic illustrations of the steps 7-15.Experimental groups: B, blank (only YES medium); C-, negative untreated control; C1 to C9: exposure solutions in increasing concentrations of the assayed compound.

Fig. 2 .
Fig. 2. Investigation of the main mechanisms of action and detoxication by efflux pumps.Schematic illustrations of the steps 19-27 for the wild type strain and two mutants of S. pombe .B, C-, C1 and C2 indicate blank, negative control and the selected concentrations of the drug, respectively.If more mutants are selected to investigate the mechanisms of action or the efflux by pumps, other plates should be filled following the same scheme.

Fig. 4 .
Fig.4.Investigation of the mechanisms of action studying the inhibition of the proliferation of the wild type , mph1 Δ, pmk1 Δ, rad3 Δ and MDR-sup strains of Schizosaccharomyces pombe .A) exposed for 20 h in liquid media to 1.5 μM and 2 μM TCS.Significant differences between the treated defective strains and their unexposed controls are indicated by * p < 0.05 and * * p < 0.01, and with the wild type treated groups are indicated by ## p < 0.01 (One-way ANOVA; Dunnett's test)[ 2 ].

Table 1
Strains of Schizosaccharomyces pombe as a predictor toxicity tool.

Table 2
Reagents for yeast growth media (YES).
* Only for agar plates.

Table 3
List of equipment and material.

Table 4
[ 6 ]ns used for the study the mechanisms of action.Extremely marked specificity and sensitivity for oxidative stress.High sensitivity to osmotic stress[ 6 ]pmk1 Δsty1 Δ